Screening cloned prokaryotic or eukaryotic hosts for protein expression and function is of great importance in the field of biotechnology. However, this technology requires evaluating each individual clone for protein expression and function. Often it is necessary to examine large numbers of cloned hosts to determine which expression system is most efficient or which protein has the characteristic function that is most desirable.
Mutagenesis is a powerful method for evaluating protein expression and function. Mutagenesis involves modifying one or more bases in a nucleotide sequence to express the protein of interest. However, because there are no reliable methods to predict the effect of modified nucleotide sequences, large numbers of point mutants and mutant proteins need to be individually evaluated.
In the past, screening individual mutants for protein expression and function was extremely time consuming and very labor intensive because individual samples needed to be evaluated. It was also very difficult to determine which point mutant expressed the protein of interest.
Proliferating cell nuclear antigen (PCNA) exemplifies this difficulty. Previously, the evaluation of PCNA point mutants was performed by testing individual clones for their effects in vitro on purified DNA polymerase .delta. (pol .delta.) using conventional protein identification and purification techniques of mutant PCNA molecules. This approach while effective, is very labor intensive. (See, for example, Refs. 9-11).
The isolation and purification of cloned proteins has been greatly facilitated by the use of affinity tags. A widely used affinity tag is a histidine (his) tag. These tags typically contain a string of four to ten consecutive histidine residues genetically engineered to be at either the amino- or carboxyl-terminus of recombinant proteins. Because his-tagged proteins bind to chelated metals attached to solid supports, they can easily be isolated by column chromatography with chelated metals attached to resins or beads as the solid support. Such chromatographic methods are referred to as Immobilized Metal Ion Affinity Chromatography (IMAC).
The use of IMAC for isolating proteins was first disclosed in Porath et al., (Ref. 31), wherein a resin was derivatized with iminodiacetic acid (IDA) and metal ions were chelated to the IDA-derivatized resin for immobilizing proteins. Columns with nickel-agarose or metal containing resin are typically used for isolating his-tagged proteins. The his-tagged protein binds to the matrix by interacting with the metal ions and is then eluted with a solution of imidazole, competing metal ions or salt. Unfortunately, the use of columns for detecting his-tagged proteins is very labor intensive and not amenable to high throughput screening.
An alternate method for identifying his-tagged proteins is disclosed in the QIAGEN product guide 1997. The QIAGEN method is a two dimensional method for detecting his-tagged proteins that uses Ni-nitrilotriacetic acid (Ni-NTA) attached to a column matrix to bind his-tagged proteins.
Similarly, a method for identifying his-tagged proteins on micro titter plates is disclosed in Paborsky et al., (Ref. 29). This method utilizes maleic anhydride-activated polystyrene microtiter plates coupled with N,N-bis[carboxymethyl]lysine, a derivative of nitrilotriacetic acid (NTA), to immobilized histidine-containing proteins. Paborsky also discloses a method for quantifying expression levels of the immobilized protein. These methods, although useful, are very labor intensive and not amenable to high throughput screening.
Based on the foregoing, there is still a great need for alternate methods and compositions for screening large numbers of cloned mutants for both protein expression and function. Methods and compositions that allow two-dimensional high throughput screening would be of particular value for designing proteins for pharmaceutical and industrial uses.